The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. We now define receptor heteromers as a macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components (1). The occurrence of receptor heteromers with different pharmacological and signaling properties opens a complete new field to search for novel drug targets useful against a variety of neuropsychiatric disorders with potentially less side effects (1-3). Our research during this last year has focused on striatal adenosine A2A receptor heteromers, which are targets for caffeine, the most consumed psychoactive drug in the world (4). Striatal adenosine A2A receptors are highly expressed in the medium spiny neurons (MSNs) of the striatal indirect pathway, where they heteromerize with dopamine D2 receptors. But striatal A2A receptors are also localized presynaptically, in glutamatergic terminals, where they heteromerize with A1 receptors and where their stimulation facilitates glutamatergic neurotransmission (5). By using integrated anatomical, electrophysiological and biochemical approaches, we found that presynaptic A2A receptors are preferentially localized in glutamatergic terminals of cortico-striatal afferents to the MSNs of the striatal direct pathway (5). This double pre- and post-synaptic segregation of striatal A2A receptors can be of pharmacological importance, since preferential postsynaptic A2A receptor antagonists should be useful in Parkinson's disease, while preferential presynaptic A2A receptor antagonists could be beneficial in dyskinetic disorders, obsessive-compulsive disorders and drug addiction. It is our working hypothesis that targeting A1-A2A and A2A-D2 receptor heteromers can provide a useful approach for targeting striatal pre- and postsynaptic A2A receptors. We have established reliable in vivo methods to analyze the function of striatal pre- and postsynaptic A2A receptors. On one hand, striatal presynaptic A2A receptor function can be analyzed by the ability of A2A receptor antagonists to block the motor output induced by cortical electrical stimulation (quantified by means of a power correlation coefficient of the input cortical electrical and output electromyographic signals) (5,6). On the other hand, striatal postsynaptic A2A receptor function can be analyzed by the ability of A2A receptor antagonists to produce locomotor activity and to block striatal ERK1/2 phosphorylation induced by cortical electrical stimulation (5,6). By screening several A2A receptor antagonists, we have already obtained evidence for a correlation between their pre- and postsynaptic activity in vivo with their relative ability to bind, in vitro, to A1-A2A and A2A-D2 receptor heteromers, respectively (submitted for publication). We have also performed studies at the protein level in order to find out specific biochemical properties of A2A receptor heteromers, which could allow their identification in native tissues and their possible selective pharmacological targeting. The approach is to find determinants of their quaternary structure and function. We previously found a selective calcium-mediated modulation of the quaternary structure and function of the A2A-D2 receptor heteromer (reviewed in ref. 7). In our most recent study, using resonance energy transfer techniques in experiments with mutated receptors, we provided for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of heteromers between adenosine A2A, cannabinoid CB1 and dopamine D2 receptors (8). In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A2A, CB1 and D2) was found to include two evolutionary conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK, signaling) of the A2A-CB1-D2 receptor heteromers. Our previously postulated presence of A1-CB1-D2 receptor heteromers in the brain (9) could then be demonstrated by analyzing MAPK signaling in striatal slices of CB1 receptor KO mice and wild-type littermates (8).